Project Summary Herpes simplex virus 1 (HSV-1) and HSV-2 cause millions of chronic infections. These viruses cause epithelial oral ?cold? sores and genital lesions, which recur lifelong due to reactivation from a latent viral reservoir in neurons. Clinical outcomes from HSV-1 infection are highly diverse, ranging from surface lesions with quite different rates of recurrence, to asymptomatic shedding, to severe and potentially lethal encephalitis. This variation is thought to be caused by a combination of factors, including viral genetic differences, human genetic predisposition, and environmental variables. Animal models serve as the cornerstone of our molecular understanding of HSV-1 disease in vivo, and provide an opportunity to dissect viral genetic contributions to pathogenesis, while controlling for host genetic factors and environmental variables. We have recently sequenced a collection of low-passage clinical isolates of HSV-1 and categorized each isolate according to its reproducible virulence phenotype in a mouse ocular model of infection. We measured virulence in this model by the virus' ability to infect, replicate, and induce lethal disease, which involved the virus initiating infection at the ocular surface, migrating to the peripheral nervous system (trigeminal ganglia), and even penetrating into the central nervous system (CNS). Using multiple statistical approaches, we found two loci in the viral tegument protein VP22 (UL49) that predictably distinguish high-virulence clinical isolates from low-virulence isolates. VP22 is a viral tegument protein that is conserved among alpha-herpesviruses, which functions as a hub of viral and cellular protein interactions. VP22 functions in close concert with the viral transactivator protein VP16 (UL48), and the viral RNAse VHS (UL41). The variant loci detected in association with murine virulence exist at a frequency of approximately 50% in the clinical HSV-1 isolates we have surveyed to date, suggesting that knowledge gleaned from these experiments will provide data on viral variants of relevance to ongoing trials for antivirals and vaccines. With increased power from additional genotype-phenotype data, we will use a genome-wide association study (GWAS) to measure the association of VP22 and other candidate loci with virulence in HSV-1. We will make recombinant HSV-1 strains to test the role(s) of VP22 and other candidate virulence loci both in vivo and in vitro. We will test the hypothesis that virulent and non-virulent phenotypes in vivo result from distinct biochemical differences in VP22 between these isolates. Data from these experiments will enable us to test the robustness of our forward genetic predictions of virulence loci in HSV-1. If these data can be linked to human impacts in the future, the ability to gauge likely virulence level based on viral genotype would provide a powerful tool for future diagnostics and prediction of clinical outcomes.